Project Summary The long-term goal of our laboratory is understand how signaling transduction by cell surface receptors is regulated. Ultimately, we would like to develop strategies for selectively activating or inhibiting these cellular activities and bypass limitations of the receptor, such as low receptor number or receptor desensitization. Every extracellular ligand (growth factor, hormone, neurotransmitter, etc) binds to a unique cell surface receptor that induces intracellular, biochemical changes that are integrated to invoke a specific change in cell physiology. While the exquisite specificity of this system has been long appreciated, the molecular mechanism by which it occurs is poorly understood. Understanding how an overlapping set of biochemical responses produces a specific physiology is the key to this problem. To better understand this important process, we are using the prototypical receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), as a model. The EGFR is critical for many developmental and homeostatic processes;stimulation of the EGFR leads to a variety of cellular changes including cell proliferation, differentiation, migration, and viability. Further, overexpression of the EGFR is associated with many cancers. We, and others, believe that the magnitude and duration of signaling to these biochemical intermediates dictates how cell physiology is altered. One way the magnitude and duration of receptor signaling is modulated is through the internalization and degradation of the receptor following ligand binding. In addition to activating intracellular signaling pathways, ligand binding also causes most cells surface receptors to internalize via clathrin-coated pits. Once inside the cell, the ligand:receptor complex moves through a series of well-defined endocytic stages until it ultimately reaches the lysosome where it undergoes degradation. It has been shown previously by a number of groups that disrupting this process can alter the effect that activated EGFRs have on the cell. However, these studies have been limited to distinguishing between cell surface and intracellular receptors. The overarching hypothesis of our research is that the endocytic pathway is a key positive and negative regulator of cell surface receptor signaling. In Aim I, we will use cultured cells and selectively disrupt EGFR trafficking through the endocytic pathway. We will assess EGFR signaling at each of these endocytic stages. Further, we will determine whether differences in signaling occur due to changes in the receptor itself, receptor:effector interactions, or the duration/magnitude of signaling. Aim II, we will build on our recent findings that spatially restricting the EGFR in cancer cells (MDA-MB-468 mammary adenocarcinoma cells) dramatically changes cell growth and viability properties. We will build on this model to determine which signals emanate from the cell surface and which are produced within the cell. Finally, in Aim III, we will explore how the endocytic pathway negatively regulates EGFR signaling. In these studies, we will determine the mechanism of signal inactivation and determine if it is unique for the receptor or effectors are inactivated in this same manner. Completion of these studies will reveal how EGFR signaling is regulated downstream of the receptor. This will guide the production of more targeted therapies to inhibit EGFR signaling (i.e. cancer) or activation of EGFR signaling (i.e. corneal wound healing).